The present invention relates to a process for the preparation of pure cyclosporin chromatographically from a mixture of different cyclosporins and other substances prepared by fermentation, using an eluent consisting essentially of high pressure carbon dioxide.
Cyclosporins are neutral, highly lipophilic, cyclic undekapeptides with a variable amino acid composition. At present, 25 different cyclosporin forms (A-Z) are known, from which the A-form has proved to be clinically the most valuable. Cyclosporins are produced by some fungi, e.g. Cylindrocarpon lucidum, Trichoderma polysporum, and various species of the genus Tolypocladium. Cyclosporins have been discovered to have not only of antibiotic activity, but also immunosuppressive properties, and they are currently used in the post-operative treatment of transplantation operations to prevent the rejection of the transplants. Cyclosporins are produced by fermenting fungal strains which produce great amounts of them. When mycclial extracts are obtained they usually contain different cyclosporin forms as a mixture. The desired form of cyclosporin has to be separated from the mixture obtained by fermentation by a purification process.
In the process currently in use, the separation of cyclosporin A is carried out by extracting the mycelial mass obtained by the fermentation first with methanol, whereafter the final separation is carried out in silica columns using different mixtures of organic solvents. The separation method is fairly slow and demands large chromatography columns with respect to the production volumes. Additionally, in a conventional process, great amounts of organic solvents must be handled, the purification and recycling of which is uneconomic and requires large equipment.
It is known that a substance which is in liquid form or in a supercritical state dissolves many compounds substantially better than the same substance in a gaseous state. Supercritical chromatography is indeed used to an ever-increasing amount to extract and separate various substances, especially in analytical applications (White et al., 1988, J. High Resolut. Chrom. & Chrom. Comm. 11:94-98).
In the U.S. Pat. No. 4,478,720, a process for fractionating of mixtures, and especially or purification of hydrocarbon mixtures by elution chromatography is disclosed. In the process, supercritical carbon dioxide without adjuvants is used as the eluent. In the international patent application WO 93/23394 optically pure S-timolol is produced from racemic R,S-timolol. Although the isomeric separation described in said application is in principle of the same kind as the separation disclosed in the present invention, the two processes differ from each other even for their grounds in that in the enantiomer separation of timolol is a question of chiral selectivity between the timolol enantiomers and the chiral column filling, whereas the present invention is based on the different polar interactions between the cyclosporins to be separated and the non-chiral column filling.
We have now found that the desired cyclosporin form can easily be obtained in pure form from a mixture of cyclosporin forms and other substances prepared by fermentation by feeding the mixture into a chromatography column with an eluent consisting of high pressure carbon dioxide and an adjuvant, and recovering from the eluent flow, the fraction which contains the desired form of cyclosporin in adequately pure form.
By the process of this invention any desired form of cyclosporin included in the starting mixture can be prepared in pure form. Preferably cyclosporin A or cyclosporin G is prepared.
The purity of cyclosporin meeting the drug requirements has to be at least 98.5 weight-%. By optimizing the process parameters cyclosporin meeting the drug requirements is directly obtained according to the process of the present invention from a certain part of the eluent flow.
An advantage of the process is in that the separation of the cyclosporin forms can be carried out continuously in phases so that the following batch of the mixture can be fed to the chromatography column soon after the previous batch, whereby the desired form of cyclosponrin meeting the drug requirements can continuously be recovered from the eluent flow coming out of the column. The purification process can thus be carried out rapidly and with small equipment with respect to the production rate.
In addition, the production process according to the invention is simple. It has only three main stages: feeding of the cyclosporin mixture to a chromatography column with an eluent consisting of carbon dioxide and an adjuvant, chromatographic separation of the desired cyclosporin form, and separation of the pure product from the eluent.
It is characteristic to the process of the invention that a mixture prepared by fermentation containing many forms of cyclosporin and other substances originating from the fermentation as extracted into a suitable solvent, e.g. toluene, is fed into the eluent flow consisting of high pressure carbon dioxide and an appropriate adjuvant. The eluent is fed into a chromatography column at a temperature of 20 to 100.degree. C., preferably at the temperature of 20 to 60.degree. C., and at an increased pressure, preferably at the pressure of 75 to 350 bar (7.5 to 35 MPa). A continuous eluent flow is running through the column. As an eluent carbon dioxide is used, to which an adjuvant or a modifying agent has been added, being e.g. a low molecular alcohol, preferably methanol or 2-propanol. A mixture of suitable low molecular alcohols can also be used. The amount of the adjuvant in the eluent is between 1 to 50, preferably 4 to 28 weight-%. The chromatography column has been packed with a solid filler, e.g. with silica particles or modified silica.
A further advantage of the process of the invention is also the fact that one can directly use as a starting material solutions which have been obtained by extracting the mycelial mass obtained by fermentation with suitable solvents, e.g. with methanol and toluene. These solutions may contain many kinds of components in addition to the cyclosporin forms to be separated. For instance pigments and other impurities originating from the cultivation are left over in the solution during the extraction. The cyclosporin mixture to be separated (mycelial extract) can further be dissolved in another solvent, e.g. toluene, dichloromethane or methanol, and this solution can be fed into the chromatography column.
An example of a solid filler which can be used is silica. The diameter of the silica particles can be from 5 to 200 .mu.m, preferably from 5 to 45 .mu.m, most preferably e.g. 10 .mu.m, and the pore size from 60 to 120 .ANG.. Examples of silica which can be used as the column filler are cyanopropyl silica and propylenediol silica. The column filler and the adjuvant of the eluent have to be selected so that the desired cyclosporin form is eluted sufficiently separated from the other forms of cyclosporin. When purifying cyclosporin A the preferable selection is silica filler together with a methanol or 2-propanol adjuvant.
In addition to the above mentioned parameters an economical realization requires the use of a correct loading ratio. The "loading ratio" of the column is the ratio (mg/g) of the total amount of cyclosporin forms fed into the column to the amount of the filling material of the column. A suitable loading ratio depends on the adjuvant on the eluent, and of the amount thereof. In a silica column, using 2-propanol adjuvant, the suitable loading ratio is e.g. from 1.5 to 4.4 mg/g. In a silica column using methanol adjuvant the suitable loading ratio is e.g. from 4.4 to 16.2 mg/g.
The eluent flow coming out from the chromatography column is monitored with a suitable detector, e.g. with an ultraviolet detector in the wave length region of 210 to 250 nm. The eluent flow coming out of the column is divided into temporally successive fractions so that the fraction containing the desired cyclosporin form is recovered. The pressure of the eluent flow fraction to be recovered is decreased, whereby the adjuvant and cyclosporin being dissolved therein are separated from the carbon dioxide, which is evaporated. The desired cyclosporin form is recovered from the adjuvant solution obtained by known processes of chemical technology.
One embodiment of the process of the invention is to perform the purification of the mixture containing the cyclosporin forms in several successive steps. In the first chromatographic purification step, a chromatography column can be packed with, for example, large silica particles. A suitable particle size may be e.g. 25 to 40 .mu.m. In the first, coarse purification silica can be loaded with a fairly large amount of the mixture to be purified. A suitable loading ratio can be, e.g., 20 mg per g of silica. From the first chromatography step, a still impure fraction is recovered which, however, contains the main part of the desired cyclosporin form.
The coarsely purified product obtained from the first purification step is injected into a second chromatography column which has been packed with a high resolution silica. In the second chromatography step, a lower loading ratio is used, as well as a column which has been packed with silica having a fairly small particle size.
A chromatographical purification with two or more steps can be more advantageous with respect to the economy of the process than a one-step purification, e.g. in such a case in which the raw material mixture to be purified contains ingredients which stain the first chromatography column.
It is important that the process parameters are optimized so that the yield of the desired product meeting the drug requirements is as high as possible. In the following Examples those conditions are given in which cyclosporin meeting the drug requirements is advantageously obtained with high yield according to the present invention. In the experiments carried out, it is shown that the adjuvants (modifying agents) obvious to a person skilled in the art in the carbon dioxide eluent are not necessarily functional, but that only certain combinations of a column filling and an adjuvant cause such a separation that industrial production will be possible. In the Examples, it is also shown that by optimizing the process parameters the problematic impurities are made to elute ahead of the product which promotes the selectivity of the separation and makes the process technically and economically feasible.